W6 Biological Control Of Bacterial Pathogens

Biological Control of Phytopathogenic Bacteria Ent 547 Fundamentals of Biological Control Fall 2005

Phytopathogenic Bacteria 

Prokaryotic    

Covalently closed circular DNA in a nucleoid. May contain plasmids. No organelles 70s ribosomes

Small, 1-10 microns x 0.5 – 1 micron.  Reproduction binary fission.  Endospores.  Entry into plant via wounds (trichome breakage, pruning, grafting, root tip elongation) or natural openings (stomata, hydathodes, lenticels). 

Phytobacterial Lifestyles 

Obligate parasites – fastidious bacteria.    



Facultative saprophytes.  



Wall-less prokaryotes. Rickettsia. Grass endophytes. Seed-borne. Prefers host but can live or survive outside host for short periods of time (1 week to 4-5 years). Seed-borne

Facultative parasites.  

Opportunistic pathogens, generally efficient pathogens once ingress is obtained. Can survive outside of host (soil) for years.

Importance of Bacteria       

Used in basic research. Industrial uses. Consumer goods (Xanthan gums, flavor, texture). Medical uses (antibiotics). Agricultural (nitrogen fixation). May be the oldest forms of life. Involved in carbon, nitrogen, and sulfur cycles.

 Cause

disease in animals, plants, and humans.

Morphology

Gram Positive Bacterial Cell Wall From Nancy Perry, University of Manchester. http://www.tea chingbiomed.man.a c.uk/student_p rojects/2001/m nlf8np2/home page.htm

Gram Negative Cell Wall

From Nancy Perry, University of Manchester. http://www.teachingbiomed.man.ac.uk/student_projects/2001/mnlf8np2/homepage.htm

Taxonomy 

Gram positive    

Bacillus Coryneform Clostridium Streptomyces



Gram negative     

 

Fastidious Phloem-limited bacteria Cell-wall free bacteria

   

Acidovorax Agrobacterium Burkholderia (Ralstonia) Erwinia Pantoea Pseudomonas Rhizomonas Xanthomonas Xylophilus

Symptoms of a Bacterial Infection in Plants 

  

 

Necrosis – dead, dying tissue margins, leaf streaks, stripes, cankers, lesions, spots, blights, vascular and pith necrosis. Chlorosis – yellow with adjacent necrotic tissue or alone. Watersoaking. Wilting – vascular occlusion from cells, gum, polysaccharide, tyloses. Soft rots – pectolytic enzymes, water release. Hyperplasia – overgrowth, galls, knots.

Signs of a Bacterial Infection Bacterial ooze or slime, especially under moist conditions.  Bacterial gum, under drier conditions.  Bacterial scale, crust, or flake under when dried.  Bacterial streaming. 

Bacterial Disease Management Resistant cultivars  Limit moisture with management  Sanitation  Antibiotics  Copper based pesticides  Bioantagonists 

Microbial Pesticides for Bacterial Disease Control Organism

Product

Target

Hosts

Formulation/ application

Agrobacterium radiobacter

Norbac 84-C Nogall Galltrol A

Crown gall

Fruit and nut trees, caneberries, roses, ornamental nursery stock

Live agar culture/water

Bacillus subtilis

Rhizo-Plus, Rhizo-Plus Konz

Streptomyces scabies

Potato

Water dispersible granule/seed treatment, soil drench, dip

Bacillus subtilis QWT713

Serenade

Erwinia amylovora (and fungi)

Stone fruits (and other crops)

Wettable powder

Pseudomonas fluorescens A506

BlightBan A506

Erwinia amylovora, frost damage

Almond, apple, apricot, blueberry, cherry, peach, pear, potato, strawberry, tomato

Wettable powder/bloom time spray

Microbial Pesticides for Bacterial Disease Control Organism

Product

Target

Hosts

Pseudomonas fluorescens

Conquer

Pseudomonas tolaasii

Mushrooms

Burkholderia solanacearum

PSSOL

Burkholderia solanacearum

Vegetables

Streptomyces lydicus

Actinovate

Soilborne fungal pathogens

Greenhouse and nursery crops, turf

Formulation/ application

Waterdispersible granule

Potential Agents 

Other Bacteria 

Wild type    



Mutants  

  

Azospirillum brasilense Other Bacillus species Streptomyces praecox Pantoea agglomerans Mutants of Burkholderia solanacearum hrp mutants

Other bacteria (mutants) Bacteriophage - bacterial viruses. Bacteriocins – small peptides that inhibit the growth of various bacteria.

Antagonism Mechanisms Antibacterial metabolites  Siderophores  Nutrient deprivation, niche exclusion  Induced resistance  Plant growth promotion 

Background Aztecs  1200 A.D.  Chinampas  Potential Biological control organisms 

  



Trichoderma spp. Pseudomonas spp. Fusarium spp.

Incorporated organic material (manure)

First Biological Control of Plant Pathogenic Bacteria 

Potato scab or common scab of potatoes  



Streptomyces scabies Streptomyces acidiscabies

Millard and Taylor, 1927  

Added green grass cuttings Added Streptomyces praecox 





Competition for active sites

Called observation “starving out”

Recent work in the 1990’s

Background 



Thus, biological control studies with bacteria has examined for over 70 years Sources of biological control bacteria     

Suppressive soils On aerial plant parts (epiphytes, phylloplane) On root surfaces (epiphytes, rhizoplane) Colonizing plant pathogens (hyperparasites) Plant disease causing bacteria (phytopathogens)

Principles  Baiting 

Schisler, D. A. and Slininger, P. J. 1994. Selection and performance of bacterial strains for biologically controlling Fusarium dry rot of potatoes incited by Gibberella pulicaris. Plant Dis. 78:251-255.

 Formulation  Mechanisms

of pathogen suppression

substrate competition and niche exclusion  siderophores  antibiotics  induced resistance (not really biological control?) 

Examples 

Products   



Agrobacterium radiobacter Bacillus subtilis Pseudomonas fluorescens – Erwinia amylovora, Pseudomonas syringae pv. syringae

Reports      

Azospirillum brasilense – root stimulant Burkholderia mutants Erwinia carotovora subsp. betavasculorum hrp- mutants Pantoea agglomerans (Erwinia herbicola) – Erwinia amylovora Bacteriophage and bacteriocins

Crown Gall 

Agrobacterium tumefaciens 





Crown gall on a wide range of dicotyledonous plants especially apple, pear, peach, cherry, almond, raspberry and roses A separate strain, biovar 3 causes crown gall of grapevine Gram negative, motile rod, related to Rhizobium

Agrobacterium tumefaciens    





Fairly ubiquitous in soil and cosmopolitan Can live saprophytically for up to two years Fairly efficient colonizer of the rhizosphere Pathogenic determinants are on the Ti (tumor-inducing) plasmid (pTi) or the Ti plasmid Are chemotactically attracted to sugars, and other root components However, A. tumefaciens strains with the Ti plasmid are more strongly attracted to wound phenolic compounds such as acetosyringone (10-7 M)

Infection



 

At greater concentrations (10-5 to 10-4 M), acetosyringone activates vir genes, these lead to the production of permeases for opine uptake, and an endonuclease that excises the T-DNA (transferred DNA) The T-DNA is released, enters and integrates into plant DNA, T-DNA codes for opines, IAA, and novel plant metabolites (agrocinopines, opines, nopalines)

Agrobacterium radiobacter: Galltrol-A, Nogall, Diegall, Norbac 84C 

Agrobacterium radiobacter strain K84   



 



Controls only nopaline producing A. tumefaciens strains This is the first biological control product for any plant disease Alan Kerr in the 1970’s

Target Pathogen/Disease: crown gall disease caused by Agrobacterium tumefaciens Crop: fruit, nut, and ornamental nursery stock Formulation: aqueous suspension containing bacterial cells, methyl cellulose, and phosphate buffer (refrigerate), agar plates, peat substrate Application: root, stem, cutting dip, or spray

Agrobacterium radiobacter K84 Similar to A. tumefaciens (same biovar) except does not have the Ti plasmid  Has pAGK84 which codes for agrocin 84 and pNOC which codes for nopaline uptake and catabolism  Mechanism of action 

 



pNOC – competition for nopaline Niche competition – efficient colonizer of roots and wound sites (chromosomal) Agrocin 84

Agrocin 84



Agrocin84 is an adenine nucleotide with a 6 glucofuran and a methylated pentamide attached (fraudulent nucleotide)

Agrocin84 

Highly selective for nopaline producing AT strains 

 

Ti plasmid of sensitive A. tumefaciens strains has NOC (nopaline catabolism) and ACC (agrocinopine catabolism) genes and permeases for uptake agrocinopene permeases imports A84 A84 blocks DNA synthesis

Luckily, the majority of AT strains are nopaline producing strains  A. radiobacter K1026 is Tra- , first genetically engineered microbe released for widespread use 

Bacillus Gram positive, soil borne, motile, endospore producing (req. oxygen), facultative anaerobe, prokaryote.  Can be found in manure and associated with plants.  There are nearly 50 species known of which only B. anthracis (anthrax) and B. cereus (food poisoning) cause disease in humans.  Known producers of bioactive metabolites act as pheromones, antibiotics, plant growth hormones, etc. 

Bacillus subtilis A13: Epic, Kodiak, Rhizo Plus, Serenade, System 3 

Bacillus subtilis A13  



Registered on peanut in 1988 Registered on cotton and broad bean in 1990

Background   



Broadbent et al., 1977 Inhibited fungi (Phytophthora spp., Pythium spp., Fusarium spp., Sclerotium spp., Rhizoctonia spp.) Stimulated growth of eggplant, dahlia and cabbage in steamed soil Seed treatment: Carrots (48%), Oats (33%), Peanuts (37%) yield increases

Kodiak Biocontrol Agent: Bacillus subtilis  Target Pathogen/Disease: Rhizoctonia solani, Fusarium spp., Alternaria spp., and Aspergillus spp. that attack roots  Crop: cotton, legumes  Formulation: dry powder; usually applied with chemical fungicides  Application: added to a slurry mix for seed treatment; hopper box treatment 

Bacillus species 

Mode of action   

Antibiosis Plant growth promotion Induced resistance Wulff et al. 2002. Biological control of black rot (Xanthomonas campestris pv. campestris) of brassicas with an antagonistic strain of Bacillus subtilis in Zimbabwe. Eur. J. Plant Pathol. 108:317-325.  Wulff et al. 2002. Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis, and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris. Plant Pathol. 51:574-584. 

Pseudomonas fluorescens BlightBan A506: Fireblight  Conquer, Victus: targets P. tolassii in mushrooms  Weller and Thomashow 

 



2-fluoroglucinol phenazine

Lindow 

Frostban

FireBlight Fireblight is caused by Erwinia amylovora  Transmitted by bees and insects to flowers  Pathogen enters flower nectaries and invades the vascular system of the plant  P. fluorescens is an effective protectant – site exclusion  Pantoea agglomerans (Erwinia herbicola) similar mechanism. 

Disease cycle of fireblight.

Burkholderia solanacearum  

Burkholderia (Pseudomonas, Ralstonia) Kempe, J. and L. Sequeira. 1983. Biological control of bacterial wilt of potatoes: attempts to induce resistance by treating tubers with bacteria. Plant Dis. 67:499-503. 



Inoculated avirulent strains of B. solanacearum, virulent but incompatible strains of B. solanacearum, and saprophytic or pathogenic pseudomonads Found   



Incompatible strain 70 (plantain) Avirulent B. solanacearum strain B82 P. fluorescens strain W163

Induced resistance

Genetic Modification of B. solanacearum Burkholderia solanacearum and many other bacterial plant pathogens have hypersensitivity and pathogenicity “clusters”  The hypersensitive reaction 



   

Rapid, localized plant cell death upon contact with a pathogen Phytoalexin accumulation Pathogenicity related protein increase Lipoxygenases increase Pathogen sequestering and death

Hrp- mutants of B. solanacearum  

Hrp = hypersensitivity pathogenicity gene cluster Mutants   



In combination with wildtype  



Decreased pathogenicity Decreased vascular spread Populations usually lower than wildtype Mutant populations are increased Wildtype populations are decreased

Mechanisms  

Competition Bacteriocin mediation?

Bacteriophage Bacteriophage are obligate intracellular viral parasites of bacteria and are compose of nucleic acids and protein  Range in size up to 200 nm long.  All have a “head” structure  Many but not all have a tail  Uses 

 

Diagnostic tool Identification and taxonomic tool

Genetic manipulation     

Loper et al. Erwinia carotovora subsp. betavasculorum Bacteriocin (phage) Out minus mutants Nearly 100% suppression of the soft rot pathogen, E. c. subsp. carotovora in potato tubers

Bacteriocins   

Most bacteriocins are proteinaceous compounds that are active again closely related bacteria There are exceptions (Agrocin 84) Reports 





Burkholderia solanacearum inhibited on plants dipped in a non-pathogenic, bacteriocin producing strain of B. solanacearum Xanthomonas campestris pv. oryzae infection incidence and severity reduced with non-pathogenic, bacteriocin producing strains. Purified bacteriocin from Pseudomonas syringae pv. ciccaronei (isol. From carob tree) – inhibited P. s. pv. savastanoi in vitro and in planta.

Summary 

Bacterial agents    



Other agents  



Bacillus Pseudomonas Burkholderia Streptomyces Bacteriophage Bacteriocins

Mechanisms  

Antibiosis Induced resistance